Nonsedating alpha-2 agonists

ABSTRACT

The present invention provides an α-2A/α-1A selective agonist that includes a compound represented by Structure 1 or a pharmaceutically acceptable salt, ester, amide, sterioisomer or racemic mixture thereof. The present invention further provides a pharmaceutical composition that contains a pharmaceutical carrier and a therapeutically effective amount of an α-2A/α-1A selective agonist that includes a compound represented by Structure 1 or a pharmaceutically acceptable salt, ester, amide, sterioisomer or racemic mixture thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/959,356 filed on Dec. 18, 2007, which is a continuation of U.S.patent application Ser. No. 11/458,731 filed on Jul. 20, 2006 now U.S.Pat. No. 7,312,238, which is a continuation of U.S. patent applicationSer. No. 10/891,953, filed Jul. 15, 2004 now U.S. Pat. No. 7,141,597,which claims benefit of priority under 35 U.S.C. §119(e) to provisionalPatent Application No. 60/502,562, filed Sep. 12, 2003, the entiredisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to molecular medicine and, moreparticularly, to α-2 adrenergic agonists that are highly selective forthe α-2A adrenergic receptor as compared to the α-1A adrenergicreceptor.

2. Background Information

A variety of conditions can be mediated, at least in part, by thesympathetic nervous system including a variety of conditions associatedwith stress. Sympathetically-enhanced conditions include, withoutlimitation, sensory hypersensitivity such as sensory hypersensitivityassociated with fibromyalgia or headache such as migraine;gastrointestinal diseases such as irritable bowel syndrome anddyspepsia; dermatological conditions such as psoriasis; cardiovasculardisorders; tachycardias; disorders of peripheral vasoconstrictionincluding Raynaud's Syndrome and scleroderma; panic attack; metabolicdisorders such as type II diabetes, insulin-resistance and obesity;disorders of muscle contraction including disorders of skeletal musclecontraction, disorders of smooth muscle contraction, spasticity, anddisorders of muscle contraction associated with tension-type headache;behavioral disorders such as, but not limited to, over-eating and drugdependence; and sexual dysfunction.

Although α-2 adrenergic agonists have shown promise in treating symptomsof sympathetically-enhanced conditions, use of these α-2 adrenergicagonists can be unsatisfactory due to concomitant sedative effects. Thissame problem limits effective α-2 adrenergic agonist treatment of otherconditions including neurological conditions, ocular conditions andchronic pain. Thus, there is a need for novel effective, non-sedatingα-2 adrenergic agonists for use as therapeutics. The present inventionsatisfies this needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides an α-2A/α-1A selective agonist thatincludes a compound represented by

or a pharmaceutically acceptable salt, ester, amide, sterioisomer orracemic mixture thereof. The present invention further provides apharmaceutical composition that contains a pharmaceutical carrier and atherapeutically effective amount of an α-2A/α-1A selective agonist thatincludes a compound represented by

(STRUCTURE 1) or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Compound 1((+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1,3-dihydro-imidazole-2-thione)from (+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1H-imidazole.

FIG. 2 shows that Compound 1 is superior to brimonidine in its abilityto alleviate sulprostone-induced tactile hypersensitivity in the absenceof sedation. The dose-responsive anti-hypersensitive and sedativeeffects of four α-2 agonists were compared in models ofsulprostone-induced tactile hypersensitivity and locomotor activity.Upper left panel: I.P. Brimonidine. Upper right panel: I.P.Dexmeditomidine. Lower left panel: Oral Compound 1. Lower right panel:I.P. Compound 2. The mean total sensitivity score and standard error ofthe mean were calculated (see solid line and solid symbols, left axis).Locomotor activity relative to vehicle-treated animals was expressed asa percentage, and the percent sedation calculated as 100% minus thepercent locomotor activity (see hatched line and open symbols, rightaxis).

DETAILED DESCRIPTION OF THE INVENTION

Adrenergic receptors mediate physiological responses to thecatecholamines, norepinephrine and epinephrine, and are members of thesuperfamily of G protein-coupled receptors having seven transmembranedomains. These receptors, which are divided pharmacologically into α-1,α-2 and β-adrenergic receptor types, are involved in diversephysiological functions including functions of the cardiovascular andcentral nervous systems. The α-adrenergic receptors mediate excitatoryand inhibitory functions: α-1 adrenergic receptors are typicallyexcitatory post-synaptic receptors which generally mediate responses inthe effector organ, while α-2 adrenergic receptors are locatedpostsynaptically as well as presynaptically, where they inhibit releaseof neurotransmitters. Agonists of α-2 adrenergic receptors currently areused clinically in the treatment of hypertension, glaucoma, spasticity,and attention-deficit disorder, in the suppression of opiate withdrawal,as adjuncts to general anesthesia and in the treatment of cancer pain.

α-2 adrenergic receptors are presently classified into three subtypesbased on their pharmacological and molecular characterization: α-2A/D(α2A in human and α-2D in rat); α-2B; and α-2C (Bylund et al.,Pharmacol. Rev. 46:121-136 (1994); and Hein and Kobilka, Neuropharmacol.34:357-366 (1995)). The α-2A and α-2B subtypes can regulate arterialcontraction in some vascular beds, and the α-2A and α-2C subtypesmediate feedback inhibition of norepinephrine release from sympatheticnerve endings. The α-2A subtype also mediates many of the centraleffects of α-2 adrenergic agonists (Calzada and Artiñano, Pharmacol.Res. 44: 195-208 (2001); Hein et al., Ann. NY Acad. Science 881:265-271(1999); and Ruffolo (Ed.), α-Adrenoreceptors: Molecular Biology,Biochemistry and Pharmacology S. Karger Publisher's Inc. Farmington,Conn. (1991)).

As disclosed herein, several α-2 agonists were assayed for α-2A/α-1Afunctional selectivity using in vitro cell-based assays. Example Idiscloses preparation of the α-2 adrenergic agonist((+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1,3-dihydro-imidazole-2-thione)from (+)-(S)-4-[1-(2,3-dimethyl-Phenyl)-ethyl]-1H-imidazole (see, also,FIG. 1). As shown in Table 1, this α-2 adrenergic agonist, denotedCompound 1, was highly α-2A/α-1A selective, as evidenced by theundetectable level of α-1A activity observed for this compound in acell-based functional assay (see, also, Example II). In contrast,dexmeditomidine was less α-2A/α-1A selective than was brimonidine (seeTable 1). These results indicate that Compound 1 is highly selective foractivation of the α-2A receptor as compared to the α-1A receptor.

TABLE 1 α-1A relative efficacy and α-1A/α-2A potency ratios of severalα-2 agonists α-1A rel. α-lA/α-2A Compound eff* potency ratio Brimonidine0.2 744 Dexmeditomidine 0.5 539 Compound 1 NA — Compound 2 0.8 980*Efficacy relative to the reference full agonist, phenylephrine. NA =not active

As further disclosed herein in Example II, the α-2A/α-1A functionalselectivity exhibited in in vitro cell-based assays correlated inverselywith in vivo sedative activity at the therapeutic dose. As revealed inFIG. 2, the α-2 agonist which was most highly selective for α-2A/α-1Afunction in vitro also exhibited the greatest separation between thetherapeutic dose which alleviated sulprostone-induced tactilesensitivity and dose resulting in significant sedation. In particular,Compound 1, administered orally at a dose of 1 μg/kg, produced a 50%reduction in sensitization (solid line, left axis), with less than 30%sedation (open diamond, right axis) at doses 100-fold and even 1000-foldgreater than the 1 μg/kg therapeutically effective dose (see FIG. 2,lower left panel). This separation between therapeutically effective andsedative doses was greater than that observed for any other α-2 agonistassayed. These results indicate that α-2A/α-1A adrenergic receptorselectivity of α-2 agonists defined using in vitro, cell-basedfunctional assays is inversely correlated with sedative activity attherapeutic doses in vivo following systemic or other peripheral dosing.These results further indicate that particularly useful α-2 agonists,with wide separation between therapeutically effective and sedativedoses, are those exhibiting α-2A/α-1A adrenergic receptor functionalselectivity.

Based on these discoveries, the present invention provides an α-2A/α-1Aselective agonist that includes a compound represented by

(STRUCTURE 1) or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof. A selective agonist of theinvention can have, for example, an α-1A efficacy less than that ofbrimonidine or a ratio of α-1A/α-2A potency greater than that ofbrimonidine. In one embodiment, an α-2A/α-1A selective agonist of theinvention includes a compound represented by FORMULA 1.

An “α-2A/α-1A selective agonist” of the invention can be characterized,in part, by (1) having greater than 25% efficacy relative to brimonidineat one or more α-2 adrenergic receptors including the α-2A adrenergicreceptor and (2) further having an α-1A efficacy less than that ofbrimonidine or a ratio of α-1A/α-2A potency greater than that ofbrimonidine. In particular embodiments, an α-2A/α-1A selective agonistof the invention has an α-1A/α-2A EC₅₀ ratio which is at least two-foldgreater than the α-1A/α-2A EC₅₀ ratio of brimonidine, or an α-1A/α-2AEC₅₀ ratio which is at least five-fold, ten-fold, twenty-fold,thirty-fold, forty-fold, fifty-fold, sixty-fold, seventy-fold,eighty-fold, ninety-fold or 100-fold greater than the α-1A/α-2A EC₅₀ratio of brimonidine. It is understood that, in addition to α-2A agonistactivity, an α-2A/α-1A selective agonist of the invention may optionallyhave agonist or antagonist activity at one or more additional adrenergicor other receptors, provided that the selective agonist satisfies thecriteria set forth above in regard to α-2A/α-1A selectivity.

Efficacy, also known as intrinsic activity, is a measure of maximalreceptor activation achieved by an agent. For the purposes ofdetermining α-2A/α-1A selectivity, efficacy is preferably determinedusing any functional assay that does not significantly amplify receptorresponse. Efficacy can be represented as a ratio or percentage of themaximal effect of the agent to the maximal effect of a standard agonistfor each receptor subtype. Brimonidine (UK14304) generally is used asthe standard agonist for the α-2A, α-2B and α-2C receptors and is usedas the standard herein where relative efficacy of an α-2 receptor isdefined. Phenylephrine is an accepted standard agonist for the α-1A,α-1B and α-1D receptors and is used herein as the standard whererelative efficacy of an α-1 receptor is defined.

In functionally characterizing an α-2A/α-1A selective agonist of theinvention, α-1A efficacy or the ratio of α-1A/α-2A potencies, or both,are compared to that of brimonidine. As used herein, the term“brimonidine” means a compound having the formula that follows

or a pharmaceutically acceptable derivative thereof. The termbrimonidine encompasses, without limitation,5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline D-tartrate (1:1),Alphagan™ and UK14304. Brimonidine, and pharmaceutically acceptablederivatives thereof can be purchased from commercial sources or preparedby routine methods, for example, as described in U.S. Pat. No.6,323,204.

Any of a variety of assays are useful to determine α-2A/α-1A functionalselectivity. As non-limiting examples, potency, activity or EC₅₀ at anα-2A receptor can be determined by assaying for inhibition of adenylatecyclase activity. Furthermore, inhibition of adenylate cyclase activitycan be assayed, without limitation, in PC12 cells stably expressing anα-2A receptor such as a human α-2A receptor. As further non-limitingexamples, potency, activity or EC₅₀ at an α-1A receptor can bedetermined by assaying for intracellular calcium. Intracellular calciumcan be assayed, without limitation, in HEK293 cells stably expressing aα-1A receptor such as a bovine α-1A receptor.

Thus, it is understood that α-2A/α-1A functional selectivity can becharacterized using any of a variety of routine functional assays, forexample, in vitro cell-based assays which measure the response of anagent proximal to receptor activation. Useful assays include, withoutlimitation, in vitro assays id such as cyclic AMP assays or GTPγSincorporation assays for analyzing function proximal to α-2 receptoractivation (Shimizu et al., J. Neurochem. 16:1609-1619 (1969); Jasper etal., Biochem. Pharmacol. 55: 1035-1043 (1998)₇ and intracellular calciumassays such as FLIPR assays and detection of calcium pulses by fluo-3for analyzing function proximal to α-1 receptor activation (Sullivan etal., Methods Mol. Biol. 114:125-133 (1999); Kao et al., J. Biol. Chem.264:8179-8184 (1989)). α-2A selectivity assays based on inhibition offorskolin-induced cAMP accumulation in PC12 cells stably expressing anα-2A receptor, and increases in intracellular calcium in HEK293 cellsstably expressing an α-1A receptor, are disclosed herein in Example IIbelow. Additional useful assays include, without limitation, inositolphosphate assays such as scintillation proximity assays (Brandish etal., Anal. Biochem. 313:311-318 (2003); assays for β-arrestin GPCRsequestration such as bioluminescence resonance energy transfer assays(Bertrand et al., J. Receptor Signal Transduc. Res. 22:533-541 (2002));and cytosensor microphysiometry assays (Neve et al., J. Biol. Chem.267:25748-25753 (1992)). These and additional assays for proximal α-2and α-1 receptor function are routine and well known in the art.

As another non-limiting example, a GTPγS assay is an assay useful fordetermining α-2A/α-1A functional selectivity. α-2 adrenergic receptorsmediate incorporation of guanosine 5′-O-(gamma-thio) triphosphate([³⁵S]GTPγS) into G-proteins in isolated membranes viareceptor-catalyzed exchange of [³⁵S]GTPγS for GDP. An assay based on[³⁵S]GTPγS incorporation can be performed essentially as described inJasper et al., supra, 1998. Briefly, confluent cells treated with anagent to be tested are harvested from tissue culture plates in phosphatebuffered saline before centrifuging at 300×g for five minutes at 4° C.The cell pellet is resuspended in cold lysis buffer (5 mM Tris/HCl, 5 mMEDTA, 0.5 mM EGTA, 0.1 mM PMSF, pH 7.5) using a Polytron Disrupter(setting #6, five seconds), and centrifuged at 34,000×g for 15 minutesat 4° C. before being resuspended in cold lysis buffer and centrifugedagain as above. Following the second wash step, aliquots of the membranepreparation are placed in membrane buffer (50 mM Tris/HCl, 1 mM EDTA, 5mM MgCl2, and 0.1 mM PMSF, pH 7.4) and frozen at −70° C. until used inthe binding assay.

GTPγS incorporation is assayed using [³⁵S]GTPγS at a specific activityof 1250 Ci/mmol. Frozen membrane aliquots are thawed and diluted inincubation buffer (50 mM Tris/HCl, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1mM DTT, 1 mM propranolol, 2 mM GDP, pH 7.4) and incubated withradioligand at a final concentration of 0.3 nM at 25° C. for 60 minutes.After incubation, samples are filtered through glass fiber filters(Whatman GF/B, pretreated with 0.5% bovine serum albumin) in a 96-wellcell harvester and rapidly washed four times with four mls of ice-coldwash buffer (50 mM Tris/HCl, 5 mM MgCl₂, 100 mM NaCl, pH 7.5). Afterbeing oven dried, the filters are transferred to scintillation vialscontaining five mls of Beckman's Ready Protein® scintillation cocktailfor counting. The EC₅₀ and maximal effect (efficacy) are then determinedfor the α-2A receptor.

It is understood that useful assays generally are performed using cellsthat naturally express significant levels of only a single α-adrenergicreceptor subtype or using transfected cells that express significantlevels of only a single recombinant α-adrenergic receptor subtype. As anon-limiting example, the adrenergic receptor can be a human receptor orhomolog thereof having a similar pharmacology. As disclosed herein,α-2A/α-1A selectivity is preferably determined with receptor-proximalassays, i.e. those in which receptor response is unamplified oramplified only minimally or those in which a rapid signal is assayed. Inview of the above, one skilled in the art will prefer to use assaysother than Receptor Selection and Amplification Technology (RSAT) assaysand similar assays in which partial and full agonism are not welldifferentiated.

A pharmaceutically acceptable salt, ester, amide, sterioisomer orracemic mixture of Compound 1 can be prepared by routine methods. Theα-2A/α-1A selective agonist shown by Structure 1 is merely exemplary ofa variety of salts, esters, amides, etc. of this compound that can bereadily prepared by one skilled in the art in a similar manner as thatdisclosed herein using well known methods of chemical synthesis,including methods similar to those exemplified herein (see Example I).

One skilled in the art understands that, in addition to the syntheticscheme shown in Example I, a variety of routes can be used to prepare,for example, the imidazole ring system of Compound 1. Such syntheses arewell known in the art, as described, for example, in Grimmett,“Imidazole and Benzimidazole Synthesis,” Ross Academic Press (1997).Furthermore, alternative routes for producing imidazole-2-thiones fromimidazoles also can be useful in preparing the selective agonist ofCompound 1. As a non-limiting example, the imidazole-2-thione ringsystem can be prepared from an imidazole ring by selectively protectingthe N1 nitrogen by a trityl group, followed by deprotonation with astrong base such as n-BuLi or LDA to form the anion at C2. The anion cansubsequently be reacted with sulfur to give the desiredimidazole-2-thione. As a further non-limiting example, an imidazole ringcan be reacted with phenylchloroformate to produce 2-imidazolone, whichcan be converted to the thione, for example, using Lawesson's reagent.These and similar methods are well known in the art for preparation ofCompound 1 and other α-2A/α-1A selective agonists of the invention.

An α-2A/α-1A selective agonist provided herein can be useful, forexample, for prevention or alleviation of a sympathetically-enhancedcondition without concomitant sedation upon peripheral administration.Any of a variety of sympathetically-enhanced conditions can be preventedor alleviated without concomitant sedation by an α-2A/α-1A selectiveagonist of the invention, including, without limitation, sensoryhypersensitivity such as that associated with fibromyalgia or headachessuch as migraines; gastrointestinal diseases such as irritable bowelsyndrome and dyspepsia; dermatological conditions such as psoriasis;cardiovascular disorders; tachycardias; disorders of peripheralvasoconstriction including Raynaud's Syndrome and scleroderma; panicattacks; metabolic disorders such as type II diabetes,insulin-resistance and obesity; disorders of muscle contractionincluding disorders of skeletal muscle contraction, disorders of smoothmuscle contraction, spasticity, and disorders of muscle contractionassociated with tension-type headache; behavioral disorders such as, butnot limited to, over-eating and drug dependence; and sexual dysfunction.

An α-2A/α-1A selective agonist provided herein also can be useful, forexample, for prevention or alleviation of chronic pain withoutconcomitant sedation upon peripheral administration. Chronic pain is aterm which means pain other than acute pain and includes, withoutlimitation, neuropathic pain, visceral pain, inflammatory pain, headachepain, muscle pain and referred pain. It is understood that chronic painis of relatively long duration, for example, several years and can becontinuous or intermittent. Chronic pain is distinguished from acutepain, which is immediate, generally high threshold, pain brought aboutby injury such as a cut, crush, burn, or by chemical stimulation such asthat experienced upon exposure to capsaicin, the active ingredient inchili peppers.

Any of a variety of types of chronic pain can be prevented or alleviatedwithout concomitant sedation by an α-2A/α-1A selective agonist of theinvention including, but not limited to, neuropathic pain such asneuropathic pain associated with diabetic neuropathy or post-herpeticneuralgia; chronic pain associated with cancer; post-operative pain;allodynic pain such as fibromyalgic pain; chronic pain associated withComplex Regional Pain Syndrome (CRPS); chronic visceral pain such asthat associated with irritable bowel syndrome or dysmennorhea; chronicheadache pain such as migraine pain, non-vascular headache pain, clusterheadache pain or daily tension headache pain; and chronic muscle painsuch as, yet not limited to, that associated with back spasm.

An α-2A/α-1A selective agonist provided herein additionally can beuseful, for example, for prevention or alleviation of a neurologicalcondition without concomitant sedation upon peripheral administration.Such a neurological condition can be, without limitation, an acute orchronic neurological condition. As non-limiting examples, acuteneurological conditions which can be prevented or alleviated withoutconcomitant sedation by an α-2A/α-1A selective agonist of the inventioninclude stroke; head and spinal cord trauma; and seizure. Furthermore,chronic neurological conditions which can be prevented or alleviatedwithout concomitant sedation by an α-2A/α1A selective agonist of theinvention include, but are not limited to, neurodegenerative diseasessuch as Alzheimer's disease; Parkinson's disease; Huntington's disease;amyotrophic lateral sclerosis and multiple sclerosis; HIV-associateddementia and neuropathy; ocular diseases such as glaucoma, diabeticneuropathy and age-related macular degeneration; and schizophrenia, drugaddiction, withdrawal and dependency, and depression and anxiety.

The term neurological condition encompasses all acute and chronicdisorders which affect, at least in part, neurons. Thus, the termneurological condition encompasses, without limitation, hypoxia-ischemia(stroke); head and spinal cord injury; epilepsy; neurodegenerativedisorders such as Alzheimer's disease, Parkinson's disease,Parkinsonism; Huntington's disease, amyotrophic lateral sclerosis andmultiple sclerosis; optic neuropathies such as glaucoma, light-inducedretinal degeneration such as photoreceptor degeneration, and maculardegeneration; disorders of photoreceptor degeneration such as retinitispigmentosa; HIV-associated dementia (acquired immunodeficiency syndromedementia complex) and HIV-associated neuropathy; metabolic,mitochondrial and infectious brain abnormalities such as, but notlimited to, encephalitis; neuropathic pain syndromes such as causalgiaor painful peripheral neuropathies; olivopontocerebellar atrophy;mitochondrial abnormalities and other biochemical disorders such asMELAS syndrome, MERRF, Leber's disease, Wernicke's encephalopathy, Rettsyndrome, homocysteinuria, hyperhomocysteinemia, hyperprolinemia,nonketotic hyperglycinemia, hydroxybutyric aminoaciduria, sulfiteoxidase deficiency, combined systems disease, lead encephalopathy;hepatic encephalopathy, Tourette's syndrome; drug addiction and drugdependency; drug withdrawal such as withdrawal from alcohol or opiates;and depression or anxiety syndromes (see, for example, Lipton andRosenberg, New Engl. J. Med. 330: 613 (1994)).

An α-2A/α-1A selective agonist provided herein further can be useful,for example, for prevention or alleviation of an ocular conditionwithout concomitant sedation upon peripheral administration. Ocularconditions to be prevented or alleviated without concomitant sedation byan α-2A/α-1A selective agonist of the invention include, withoutlimitation, glaucoma; macular degeneration; and retinopathies such asdiabetic retinopathy.

Any of a variety of ocular conditions can be prevented or alleviatedwithout concomitant sedation following peripheral administration of anα-2A/α-1A selective agonist of the invention. Such conditions include,yet are not limited to, diabetic retinopathy; macular edema such as thatassociated with diabetes; conditions of retinal degeneration such asglaucoma, macular degeneration such as age-related macular degeneration(ARM) and retinitis pigmentosa; retinal dystrophies; inflammatorydisorders of the retina; vascular occlusive conditions of the retinasuch as retinal vein occlusions or branch or central retinal arteryocclusions; retinopathy of prematurity; retinopathy associated withblood disorders such as sickle cell anemia; elevated intraocularpressure; ocular itch; damage following retinal detachment; damage orinsult due to vitrectomy, retinal or other surgery; and other retinaldamage including therapeutic damage such as that resulting from lasertreatment of the retina, for example, pan-retinal photocoagulation fordiabetic retinopathy or photodynamic therapy of the retina. Ocularconditions that can be prevented or alleviated without concomitantsedation by peripheral administration of an α-2A/α-1A selective agonistof the invention further include, without limitation, genetic andacquired optic neuropathies such as optic neuropathies characterizedprimarily by loss of central vision, for example, Leber's hereditaryoptic neuropathy (LHON), autosomal dominant optic atrophy (Kjer disease)and other optic neuropathies such as those involving mitochondrialdefects, aberrant dynamin-related proteins or inappropriate apoptosis;and optic neuritis such as that associated with multiple sclerosis,retinal vein occlusions or photodynamic or laser therapy. See, forexample, Carelli et al., Neurochem. Intl. 40:573-584 (2002); and Olichonet al., J. Biol. Chem. 278:7743-7746 (2003). It is understood that theseand other ocular abnormalities, especially those of the neurosensoryretina, can be prevented or alleviated without concomitant sedationusing the selective agonists of the invention.

In addition to preventing or alleviating sympathetically-enhancedconditions, neurological conditions, ocular conditions and chronic pain,an α-2A/α-1A selective agonist can be useful for preventing oralleviating other disorders without concomitant sedation. Such adisorder can be, for example, attention deficit disorder (ADHD/ADD),which is a disorder primarily characterized by inattention,distractibility and impulsiveness starting before the age of seven.Symptoms can include, without limitation, fidgeting and squirming,difficulty in remaining seated, easy distractibility, difficultyawaiting one's turn, difficulty in refraining from blurting out answers,inability to follow instructions, excessive talking, and otherdisruptive behavior (Anderson, supra, 1994). Furthermore, whileoriginally recognized in children, ADHD/ADD continues into adulthood inmany individuals (see, for example, Block, Pediatr. Clin. North Am.45:1053-1083 (1998); and Pary et al., Ann. Clin. Psychiatry 14:105-111(2002)). One skilled in the art understands that a method of theinvention can be useful for preventing or alleviating ADHD/ADD inchildren and adults having mild as well as severe forms of the disorder.

An α-2A/α-1A selective agonist also can be useful to prevent oralleviate nasal congestion; diarrhea; urinary disorders such ashyperactive micturition and overactive bladder; congestive heartfailure; or a psychosis such as a manic disorder. Furthermore, anα-2A/α-1A selective agonist can be useful to prevent or alleviate one ormore symptoms associated with anesthesia such as nausea, vomiting,shivering or panic; or to enhance memory and cognitive processes,without concomitant sedation.

As disclosed herein, an α-2A/α-1A selective agonist of the invention ischaracterized, in part, by the ability to prevent or alleviate any of avariety of sympathetically-enhanced conditions, neurological conditions,ocular conditions and types of chronic pain without concomitantsedation. The term “alleviating,” as used herein, means reducing by atleast about 50% at least one symptom of the particular condition or typeof chronic pain being treated.

As is well known in the art, sedation is a term that means a reductionin motor activity. The phrase “without concomitant sedation,” as usedherein in reference to a selective agonist, means that, upon peripheraladministration, the selective agonist produces less than about 30%sedation at a dose 10-fold greater than the dose of selective agonistrequired to produce a 50% reduction of one or more symptoms of theparticular condition or type of chronic pain being treated. For example,as shown in FIG. 2 (lower left panel), Compound 1 was administeredorally at a dose of 1 μg/kg to produce a 50% reduction in sensitizationscore (solid line, left axis) with less than 30% sedation (open diamond,right axis) at doses 100-fold and even 1000-fold greater than the 1μg/kg therapeutically effective dose. Thus, the α-2A/α-1A selectiveagonist represented by formula Compound 1 has effective therapeuticactivity “without concomitant sedation.” In contrast, many α-2 agonistssuch as dexmeditomidine are completely sedating at doses 10-fold greaterthan the dose required to produce a 50% reduction in sensitizationscore.

As non-limiting examples, the dose of α-2A/α-1A selective agonistrequired to produce about 30% sedation (reduction in motor activity) canbe at least 25-fold greater than, 50-fold greater than, 100-fold greaterthan, 250-fold greater than, 500-fold greater than, 1000-fold greaterthan, 2500-fold greater than, 5000-fold greater than, or 10,000-foldgreater than the dose required to produce a 50% reduction in one or moresymptoms of the particular condition or type of chronic pain beingtreated. Methods for determining the extent of a reduction in a symptomas well as the extent of sedation are described herein and further arewell known in the art.

The present invention further provides a pharmaceutical composition thatcontains a pharmaceutical carrier and a therapeutically effective amountof an α-2A/α-1A selective agonist that includes a compound representedby

(STRUCTURE 1) or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof. In a pharmaceutical compositionof the invention, the selective agonist can have, for example, an α-1Aefficacy less than that of brimonidine or a ratio of α-1A/α-2A potencygreater than that of brimonidine. In one embodiment, the α-2A/α-1Aselective agonist of the invention included in a pharmaceuticalcomposition of the invention contains a compound represented byStructure 1.

Thus, the invention provides a pharmaceutical composition containing aneffective amount of a pharmaceutical carrier and a therapeuticallyeffective amount of an α-2A/α-1A selective agonist of the invention.Such a pharmaceutical composition can be useful for preventing oralleviating, for example, any of the sympathetically-enhanced,neurological, or ocular conditions or types of chronic pain disclosedherein above without concomitant sedation. A pharmaceutical compositionof the invention includes an α-2A/α-1A selective agonist and furtherincludes a pharmaceutically acceptable carrier, which is any carrier,excipient or diluent that has substantially no long term or permanentdetrimental effect when administered to a subject. An excipientgenerally is mixed with an active α-2A/α-1A selective agonist, orpermitted to dilute or enclose the selective agonist. A carrier can be asolid, semi-solid, or liquid agent that acts as an excipient or vehiclefor the active selective agonist. Examples of solid carriers useful inthe pharmaceutical compositions of the invention include, withoutlimitation, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, polyalkylene glycols, talcum,cellulose, glucose, sucrose and magnesium carbonate. Suppositoryformulations can include, for example, propylene glycol as a carrier.Examples of pharmaceutically acceptable carriers further include,without limitation, water, such as distilled or deionized water; saline;aqueous dextrose, glycerol, ethanol and the like. It is understood thatthe active ingredients within a pharmaceutical composition can besoluble or can be delivered as a suspension in the desired carrier ordiluent.

A pharmaceutical composition can optionally include one or more agentssuch as, without limitation, emulsifying agents, wetting agents,sweetening or flavoring agents, tonicity adjusters, preservatives,buffers or anti-oxidants. Tonicity adjustors useful in a pharmaceuticalcomposition of the invention include, but are not limited to, salts suchas sodium acetate, sodium chloride, potassium chloride, mannitol orglycerin and other pharmaceutically acceptable tonicity adjustors.Preservatives useful in the pharmaceutical compositions of the inventioninclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. Variousbuffers and means for adjusting pH can be used to prepare apharmaceutical composition, including, but not limited to, acetatebuffers, citrate buffers, phosphate buffers and borate buffets.Similarly, anti-oxidants useful in pharmaceutical compositions are wellknown in the art and include, for example, sodium metabisulfite, sodiumthiosulfate, acetylcysteine, butylated hydroxyanisole and butylatedhydroxytoluene. It is understood that these and other substances knownin the art of pharmacology can be included in a pharmaceuticalcomposition of the invention. See, for example, Remington'sPharmaceutical Sciences Mack Publishing Company, Easton, Pa. 16thEdition 1980. It is further understood that a pharmaceutical compositioncontaining an α-2A/α-1A selective agonist can optionally be administeredin conjunction with one or more other therapeutic substances, in thesame or a different pharmaceutical composition and by the same ordifferent routes of administration.

An α-2A/α-1A selective agonist is peripherally administered to a subjectin a therapeutically effective amount. Such a therapeutically effectiveamount generally is the minimum dose necessary to achieve the desiredprevention or alleviation of one or more symptoms of, for example, asympathetically-enhanced condition, neurological condition, ocularcondition or chronic pain, such as that amount roughly necessary toreduce to tolerable levels the discomfort caused by thesympathetically-enhanced condition, neurological condition, ocularcondition or chronic pain. Such a dose can be an amount which reduces atleast one symptom of the condition or type of pain by at least about 50%and generally is in the range of 0.1-1000 mg/day and can be, forexample, in the range of 0.1-500 mg/day, 0.5-500 mg/day, 0.5-100 mg/day,0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5 mg/day, with theactual amount to be administered determined by a physician taking intoaccount the relevant circumstances including the severity and type ofsympathetically-enhanced condition, neurological condition, ocularcondition or chronic pain; the age and weight of the subject; thesubject's general physical condition; and the pharmaceutical formulationand route of administration. As discussed further below, apharmaceutical composition of the invention also can be useful in theform of a suppository or extended release formulation such as, withoutlimitation, a dermal patch, formulation for deposit on or under theskin, or formulation for intramuscular injection.

In one embodiment, a pharmaceutical composition of the invention is anophthalmic composition. An ophthalmic composition contains anophthalmically acceptable carrier, which is any carrier that hassubstantially no long term or permanent detrimental effect on the eye towhich it is administered. Examples of ophthalmically acceptable carriersinclude, without limitation, water, such as distilled or deionizedwater; saline; and other aqueous media. Ophthalmic compositions canincorporate, for example, soluble α-2A/α-1A selective agonist, or anα-2A/α-1A selective agonist as a suspension in a suitable carrier.

Topical ophthalmic compositions also are useful. Such compositionsencompass, without limitation, ocular drops, ocular ointments, oculargels and ocular creams. Such ophthalmic compositions are easy to applyand deliver the selective agonist effectively. Components of anon-limiting, exemplary topical ophthalmic composition are shown belowin Table 2.

TABLE 2 Ingredient Amount (% W/V) Compound 1 about 0.0001 to about 0.1Preservative   0-0.10 Vehicle 0-40 Tonicity Adjustor 1-10 Buffer0.01-10   pH Adjustor q.s. pH 4.5-7.5 antioxidant As needed PurifiedWater As needed to make 100%

A preservative can be included, if desired, in an ophthalmic compositionof the invention. Such a preservative can be, without limitation,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuricacetate, or phenylmercuric nitrate. Vehicles useful in a topicalophthalmic composition include, yet are not limited to, polyvinylalcohol, povidone, hydroxypropyl methyl cellulose, poloxamers,carboxymethyl cellulose, hydroxyethyl cellulose and purified water.

A tonicity adjustor also can be included, if desired, in an ophthalmiccomposition of the invention. Such a tonicity adjustor can be, withoutlimitation, a salt such as sodium chloride, potassium chloride, mannitolor glycerin, or another pharmaceutically or ophthalmically acceptabletonicity adjustor.

Various buffers and means for adjusting pH can be used to prepareanophthalmic composition in the invention, provided that the resultingpreparation is ophthalmically acceptable. Such buffers include, but arenot limited to, acetate buffers, citrate buffers, phosphate buffers andborate buffers. It is understood that acids or bases can be used toadjust the pH of the composition as needed. Ophthalmically acceptableantioxidants useful in preparing an ophthalmic composition include, yetare not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

An α-2A/α-1A selective agonist of the invention or a pharmaceuticalcomposition containing such a selective agonist is peripherallyadministered to a subject. As used herein in reference to an α-2A/α-1Aselective agonist, the term “peripherally administering” or “peripheraladministration” means introducing the α-2A/α-1A selective agonist into asubject outside of the central nervous system. Thus, peripheraladministration encompasses any route of administration other than directadministration to the spine or brain.

A therapeutically effective amount of an α-2A/α-1A selective agonist canbe peripherally administered to a subject by any of a variety of meansdepending, for example, on the type of condition or chronic pain to beprevented or alleviated, the pharmaceutical formulation, and thehistory, risk factors and symptoms of the subject. Suitable routes ofperipheral administration include both systemic and localadministration. As non-limiting examples, a therapeutically effectiveamount of an α-2A/α-1A selective agonist can be administered orally;parenterally; by subcutaneous pump; by dermal patch; by intravenous,intra-articular, subcutaneous or intramuscular injection; by topicaldrops, creams, gels or ointments; as an implanted or injected extendedrelease formulation; or by subcutaneous minipump or other implanteddevice.

One skilled in the art understands that peripheral administration can belocal or systemic. Local administration results in significantly more ofan α-2A/α-1A selective agonist being delivered to and about the site oflocal administration than to regions distal to the site ofadministration. Systemic administration results in delivery of anα-2A/α-1A selective agonist essentially throughout at least the entireperipheral system of the subject.

Routes of peripheral administration useful for delivery of an α-2A/α-1Aselective agonist or pharmaceutical composition of the inventionencompass, without limitation, oral administration, topicaladministration, intravenous or other injection, and implanted minipumpsor other extended release devices or formulations. An α-2A/α-1Aselective agonist or pharmaceutical composition of the invention can beperipherally administered, without limitation, orally in any acceptableform such as in a tablet, pill, capsule, powder, liquid, suspension,emulsion or the like; as an aerosol; as a suppository; by intravenous,intraperitoneal, intramuscular, subcutaneous or parenteral injection; bytransdermal diffusion or electrophoresis; topically in any acceptableform such as in drops, creams, gels or ointments; and by minipump orother implanted extended release device or formulation. An α-2A/α-1Aselective agonist optionally can be packaged in unit dosage formsuitable for single administration of precise dosages, or in sustainedrelease dosage form for continuous controlled administration.

Chronic administration of an α-2A/α-1A selective agonist orpharmaceutical composition of the invention can be useful, for example,for prevention or alleviation of chronic pain or another chroniccondition such as, without limitation, a chronic neurological condition.Means for repeated or continuous peripheral administration include,without limitation, repeated oral or topical administration, andadministration via subcutaneous minipump. As non-limiting examples, anα-2A/α-1A selective agonist or pharmaceutical composition of theinvention can be peripherally and chronically administered by continuousintravenous administration via implanted infusion minipump, or using anextended release formulation.

It is understood that slow-release formulations can be useful forpreventing or alleviating chronic pain or another chronic condition suchas, without limitation, a chronic neurodegenerative condition. It isfurther understood that the frequency and duration of dosing of such aslow-release formulation will be dependent, in part, on the preventionor extent of alleviation desired and the half-life of the α-2A/α-1Aselective agonist, and that a variety of routes of administration areuseful for delivering slow-release formulations, as discussedhereinabove.

An α-2A/α-1A selective agonist or ophthalmic composition of theinvention can be peripherally administered to a subject to prevent oralleviate an ocular condition by any of a variety of means depending, inpart, on the characteristics of the selective agonist to be administeredand the history, risk factors and symptoms of the subject. Peripheralroutes of administration suitable for preventing or alleviating anocular condition include both systemic and local administration. Inparticular embodiments, an ophthalmic composition containing anα-2A/α-1A selective agonist is administered topically such as by oculardrops, or by local injection, or is released from an intraocular orperiocular implant.

Systemic and local routes of administration useful in preventing oralleviating an ocular condition by administration of an α-2A/α-1Aselective agonist or ophthalmic composition of the invention encompass,without limitation, oral gavage; intravenous injection; intraperitonealinjection; intramuscular injection; subcutaneous injection; transdermaldiffusion and electrophoresis; topical eye drops and ointments;periocular and intraocular injection including subconjunctivalinjection; extended release delivery devices such as locally implantedextended release devices; and intraocular and periocular implantsincluding bioerodible and reservoir-based implants.

In one embodiment, an ophthalmic composition containing an α-2A/α-1Aselective agonist is administered topically to the eye. The α-2A/α-1Aselective agonist can be administered, for example, as part of anophthalmic solution such as ocular drops. In another embodiment, anophthalmic composition containing an α-2A/α-1A selective agonist of theinvention is injected directly into the eye. In a further embodiment, anophthalmic composition containing an α-2A/α-1A selective agonist of theinvention is released from an intraocular or periocular implant such asa bioerodible or reservoir-based implant.

As indicated above, an ophthalmic composition containing an α-2A/α-1Aselective agonist can be administered locally via an intraocular orperiocular implant, which can be, without limitation, bioerodible orreservoir-based. As used herein, the term “implant” refers to anymaterial that does not significantly migrate from the insertion sitefollowing implantation. An implant can be biodegradable,non-biodegradable, or composed of both biodegradable andnon-biodegradable materials; a non-biodegradable implant can include, ifdesired, a refillable reservoir. Implants useful for preventing oralleviating an ocular condition include, for example, patches,particles, sheets, plaques, microcapsules and the like, and can be ofany shape and size compatible with the selected site of insertion, whichcan be, without limitation, the posterior chamber, anterior chamber,suprachoroid or subconjunctiva of the eye. It is understood that auseful implant generally releases the implanted ophthalmic compositionat a therapeutically effective dose to the eye of the subject over anextended period of time. A variety of ocular implants and extendedrelease formulations suitable for ocular release are well known in theart, as described, for example, in U.S. Pat. Nos. 5,869,079 and5,443,505.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Preparation of Compound 1

This example describes preparation of the α-2A/α-1A selective agonist,Compound 1.

A. Preparation of Compound 1((+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1,3-dihydro-imidazole-2-thione)

A mixture of (+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1H-imidazole(dexmeditomidine; 2.00° g, 10.0 mmol) prepared as described in Cordi etal., Synth. Comm. 26: 1585 (1996), in THF (45 mL) and water (40 mL) wastreated with NaHCO₃ (8.4 g, 100 mmol) and phenylchlorothionoformate (3.7mL, 27.4 mmol). After stirring for four hours at room temperature, themixture was diluted with water (30 ml) and ether (75 mL). The organiclayer was removed, and the aqueous layer extracted twice with a 50 mlvolume of ether. The organic layers were dried over MgSO₄ and filtered.The residue was concentrated under vacuum, diluted with MeOH (54 mL) andreacted with NEt₃ (6.5 mL) at room temperature for 16 hours. The solventwas removed under vacuum and replaced with 30% CH₂C₁₂:hexane. Thesolvent was removed again and solids formed. After further resuspensionin 30% CH₂C₁₂:hexane, the solid was collected on a filter, washed withCH₂C₁₂:hexane and dried under vacuum to give Compound 1((+)-(S)-4-[1-(2,3-dimethyl-phenyl)-ethyl]-1,3-dihydro-imidazole-2-thione)1.23 g (53%). A schematic of the preparation of Compound 1 is shownabove.

Characterization of the product yielded the following. Optical rotation:[a]_(D)20+14° (c 1.25 in MeOH). 1 ^(H) NMR: (300 MHz, DMSO) d 11.8 (s,1H), 11.6 (s, 1H), 7.03-7.01 (m, 2H), 6.95-6.91 (m, 1H), 6.50 (s, 1H),4.15 (q, J=6.9 Hz, 1H), 2.25 (s, 3H), 2.20 (s, 3H), 1.38 (d, J=6.9 Hz,3H).

B. Procedure for the preparation of Compound 2(5-(1H-Imidazol-4-ylmethyl)-cyclohex-1-enyl]-methanol)

8-(2-Benzyloxy-ethyl)-1,4-dioxa-spiro[4.5]decane (Intermediate R1; 1.02g, 3.70 mmol) was prepared as described in Ciufolini et al., J. Amer.Chem. Soc. 113: 8016 (1991). This compound was dissolved in acetone (100mL): H₂O (5 mL) and reacted with TsOH (140 mg, 0.74 mmol) at 45° C. for5 hours. After a standard aqueous work-up the material was purified bychromatography on SiO₂ to give 4-(2-benzyloxy-ethyl)-cyclohexanone as acolorless oil (97%).

A solution of LDA (33 ml, 1.5 M in Et₂O) in THF (50 mL) at −78° C. wastreated with 4-(2-benzyloxy-ethyl)-cyclohexanone (9.5 g, 40.2 mmol). Themixture was warmed to 0° C. over 30 minutes before re-cooling to −78° C.and adding HMPA (7 mL). Methyl cyanoformate (4.1 mL, 85 mmol) was added,and the mixture stirred for 15′ minutes before aqueous quench andwork-up. The product was purified by chromatography on SiO₂ with 10%EtOAc:Hx. 5-(2-Benzyloxy-ethyl)-2-oxo-cyclohexanecarboxylic acid methylester was isolated, 5.8 g (49%), and reduced with an equivalent of NaBH₄in MeOH at −10° C. The alcohol (Intermediate R2 above) was purified bychromatography on SiO₂ with 30 to 50% EtOAC:Hx. (−90% yield).

A solution of 5-(2-benzyloxy-ethyl)-2-hydroxy-cyclohexanecarboxylic acidmethyl ester (Intermediate R2; 0.72 g, 2.48 mmol) in pyridine (10 mL)was treated with SOCl₂ (0.73 mL, 12.4 mmol) at −20° C. The mixture wasallowed to react for 15 minutes and was then warmed to 55° C. for 16hours. The solvents were removed under vacuum and the residue wasdiluted in ether at 0° C. The solution was quenched with water, washedwith 1M HCl, 5% NaOH and brine. The organic material was dried overMgSO₄, filtered and freed of solvent. The mixture was diluted withbenzene, and water was removed by azeotropic distillation under vacuum.The residue was dissolved in benzene (15 mL), and DBU (0.76 mL, 5 mmol)was added. The mixture was reacted for 30 minutes at room temperature.After work-up and chromatography on SiO₂, with 20% EtOAc:Hx,5-(2-benzyloxy-ethyl)-cyclohex-1-enecarboxylic acid methyl ester(Intermediate R3) was isolated (0.56 g (82%)).

Intermediate R3 was dissolved in THF (100 mL) and added to a solution ofDIBAL (70 mL, 1M in hexanes) in THF (160 mL) at −35° C. for 35 minutes.The mixture was quenched with Rochelle's salt solution, and extractedwith ether. The dried residue was purified by chromatography on SiO₂with 30% EtOAc:Hx to yield[5-(2-benzyloxy-ethyl)-cyclohex-1-enyl]-methanol 4.6 g (80%). A solutionof the alcohol (4.0 g, 18.7 mmol) in DMF (60 mL) was treated withtriethylamine (3 mL) followed by TBSCl (3.0 g, 22.4 mol) for 20 minutesat room temperature. The residue was isolated from an aqueous work-upand purified by chromatography to give[5-(2-benzyloxy-ethyl)-cyclohex-1-enylmethoxy]-tert-butyl-dimethyl-silane(Intermediate R4) 3.6 g (63%).

The benzyl protected alcohol (Intermediate R4) (2.0 g, 5.55 mmol) in THF(20 mL) was cooled to −70° C., and NH₃ was condensed in this flask (−20mL). Na chunks were added, and the mixture was allowed to stir at −70°C. for 15 minutes. The mixture was warmed to −30° C. for 20 minutes,quenched with NH₄Cl, and isolated by extraction. The residue waspurified by chromatography on SiO₂ with 25% EtOAc:Hx (99%). The alcoholwas oxidized by the standard “Swern” protocol. The alcohol2-[3-(tert-butyl-dimethyl-silanyloxymethyl)-cyclohex-3-enyl]-ethanol(1.3 g, 4.8 mmol) was added to a solution of oxalyl chloride (3.55 mL,7.1 mmol) in CH₂Cl₂ (30 mL) with DMSO (0.63 mL, 8.9 mmol) at −78° C.After 40 minutes, NEt₃ (2.51 mL) was added, and the mixture was warmedto room temperature. After standard aqueous work-up and purification,[3-(tert-butyl-dimethyl-silanyloxymethyl)-cyclohex-3-enyl]-acetaldehyde(Intermediate R5) was isolated (˜95%).

The following preparation followed the procedure by Horne et al.,Heterocycles 39:139 (1994). A solution of the aldehyde (Intermediate R5;0.34 g, 1.3 mmol) in EtOH (5 mL) was treated with tosylmethyl isocyanide(TosMIC; Aldrich; 0.25 g; 1.3 mmol) and NaCN (˜15 mg, cat) and allowedto stir at room temperature for 20 minutes. The solvent was removed invacuo; the residue was dissolved in ˜ 7M NH₃ in MeOH and transferred toa resealable tube before heating at 100° C. for 15 hours. The mixturewas concentrated and purified by chromatography on SiO₂ with 5% MeOH(sat. w/NH₃):CH₂Cl₂. A solution of the product in THF with TBAF (1.5eq.) was stirred at room temperature after aqueous workup. The crudeproduct was chromatographed (5-7% NH₃/MeOH in CH₂C₁₂) and designatedCompound 2.

Characterization of Compound 2 yielded the following. ¹H NMR (300 MHz,DMSO-d⁶) d 7.52 (s, 1H), 6.72 (s, 1H), 5.54 (brs, 1H), 3.73 (s, 2H),2.46 (d, J=6 Hz, 2H), 1.5-2.1 (m, 6H), 1.0-1.55 (m, 1H)

Example II Characterization of an α-2 Agonist with Greater α-2A/α-1AFunctional Selectivity than Brimonidine

This example demonstrates that α-2A/α-1A selectivity in receptorproximal functional assays correlates with non-sedating in vivoactivity.

A. In Vitro Functional Assays

Proximal functional activity at the α-1A and α-2A adrenergic receptorswas compared for brimonidine, dexmeditomidine, Compound 1 and Compound2. Brimonidine was obtained from Sigma; dexmeditomidine was prepared asdescribed in Cordi et al., supra, 1996; and Compounds 1 and 2 weresynthesized as described in Example I above. The α-adrenergic receptorpharmacological profiles were analyzed in assays using cell lines stablyexpressing α-2A or α-1A receptors, described below.

To assess α-1A activity, compounds were functionally tested for theability to stimulate an increase in intracellular calcium in HEK293cells stably expressing bovine α-1A receptor. α-1A relative efficacy wasdetermined in reference to the full agonist, phenylephrine, as describedbelow. As summarized in Table 1 shown above, dexmeditomidine andCompound 2 had α-1A relative efficacies greater than that ofbrimonidine, while the α-1A relative efficacy of Compound 1 was so lowas to be undetectable in this assay.

The same compounds were also functionally assayed for proximal α-2Afunction by assaying for inhibition of forskolin-induced cAMPaccumulation in PC12 cells stably expressing human α-2A receptor.Intracellular cAMP levels were determined using the Biotrak cAMP enzymeimmunoassay system described below. The EC₅₀ for α-2A cAMP inhibitionwas expression as a ratio with the α-1A EC₅₀ to give an α-1A/α-2Apotency ratio. As shown in Table 1 above, the α-2 adrenergic agonistdenoted Compound 1 was highly α-2A/α-1A selective, as evidenced by theundetectable level of α-1A activity observed for this compound. Incontrast, dexmeditomidine, for example, was less α-2A/α-1A selectivethan was brimonidine. These results indicate that Compound 1 is highlyselective for activation of the α-2A′ receptor as compared to the α-1Areceptor.

Stable cell lines expressing an adrenergic receptor were established asfollows. The bovine α-1A, hamster α-1B, human α-2A and human α-2Creceptor cDNAs were blunt-end subcloned into the NheI-EcoRI sites in theretroviral vector pCL BABE Puro. The retroviral constructs were verifiedby double stranded DNA sequencing. High titer pseudotyped retroviralparticles were produced by co-transfecting HEK293GP, a HEK293 cell linestably expressing Gag-Pol of the Maloney leukemia virus, with theappropriate retroviral vector and pMD.G, an expression vector for thevesicular stomatitis virus envelope protein, VSV-G. Sixteen hours aftertransfection, the media (DMEM, 10% FCS) was changed; the high titer(˜1×106 pfu/mL) media was then harvested forty-eight hours later. Thesupernatant was filtered through a 0.4 μM filter.

The human α-2A receptor supernatant was added, in varying amounts, tonaive PC12 cells, which were then incubated for 48 hours. The transducedcell population was replated at a lower density and grown in mediacontaining 100 μg/ml puromycin. Non-transduced cells were killed withinthree days, and single foci grew within two months. The foci werepicked, expanded, and assayed for receptor density by brimonidineradioligand binding. Functional α-2 receptor activity was confirmed byinhibition of forskolin-induced cAMP accumulation.

The bovine α-1A receptor supernatant was added, in varying amounts, tonaive HEK293 cells, which were then incubated for 48 hours. Thetransduced cell population was replated at a lower density and grown inmedia containing 0.25 μg/ml puromycin. Significant cell death wasevident within three days, with single foci appearing within two weeks.After the foci were picked and expanded, subclones were functionallyassayed for α-1 receptor expression by measuring phenylephrine-inducedintracellular Ca₊₂ accumulation as described below. Receptor density wasmeasured in a prazosin radioligand binding assay.

Intracellular Ca₊₂ responses were measured in HEK293 cells stablyexpressing the bovine α-1A adrenergic receptor. Between 40,000 to 50,000cells were plated per well in 96-well poly-D-lysine coated plates in 0.2ml DMEM containing 10% heat-inactivated fetal calf serum, 1%antibiotic-antimycotic and 0.25 g/ml puromycin one day prior to use.Cells were washed twice with HBSS supplemented with 10 mM HEPES, 2.0 mMCaCl₂ and 2.5 mM probenicid, and subsequently incubated at 37° C. for 60minutes with 4 M Fluo-4 (Molecular Probes; Eugene, Oreg.). Extracellulardye was washed from the plates twice prior to placing the plates in thefluorometric imaging plate reader (FLIPR; Molecular Devices; Sunnyvale,Calif.). Compounds to be assayed were diluted in HBSS and aliquoted intoa 96-well microplate; compounds were tested over the concentration rangeof 0.64 nM to 10,000 nM. Data for Ca₊₂ responses, were obtained inarbitrary fluorescence units.

The percent α-1A efficacy (% E) was determined by comparing the maximumeffect of each agonist to the maximum effect of the standard fullagonist phenylephrine. The values represent the mean and SEM from 3-15independent experiments. The fold-selectivity of the agonists for α-2receptors relative to α-1 receptors was calculated from the ratio oftheir mean EC₅₀s for activating the α-1A and α-2A receptors.

Intracellular cAMP measurement was performed as follows. PC12 cellsstably expressing the human α-2A adrenergic receptor were plated in96-well poly-D-lysine coated plates at a density of 30,000 cells perwell in 100 μl DMEM supplemented with 10% horse serum, 5% heatinactivated fetal bovine serum, 1% antibiotic-antimycotic and 100 μg/mlpuromycin. Cells were grown overnight at 37° C. and 5% CO₂. Cells weredosed by adding an equal volume of media containing IBMX to a finalconcentration of 1 mM), forskolin (to a final concentration of 10 M) andthe appropriate drug dilution (to a final concentration of between 10-5Mand 10-12 M). After a 10 minute incubation, media was aspirated, and thecells lysed with 200 μl lysis buffer (Amersham Biosciences; Piscataway,N.J.). Plates were stored at −20° C. for up to 24 hours prior to assay.Intracellular cAMP was determined using the Biotrak cAMP enzymeimmunoassay system (Amersham Biosciences) according to themanufacturer's instructions. Plates were read on a plate reader at 450nm.

Dose response curves for in vitro assays were generated usingKaleidaGraph (Synergy Software; Reading, Pa.) by least squares fits tothe equation, response=maximum response+((minimum response−maximumresponse)/(1+ (concentration of ligand/EC₅₀)). The percent α-1A efficacywas determined by comparing the maximum effect of the compound to theeffect of the standard full agonist phenylephrine.

B. In Vivo Efficacy and Sedative Effects

In addition to the cell-based assays described above, the various α-2agonists were assayed for the ability to alleviate sulprostone-inducedtactile hypersensitivity and for sedating activity at various doses. Thetactile hypersensitivity of 5-6 mice per group was scored every fiveminutes between 15 and 50 minutes following intraperitoneal dosing.Vehicle treated animals typically had a score of about 4. In addition,the locomoter activity of 5-6 mice per group was measured in a fiveminute period 30 minutes following intraperitoneal dosing. Locomoteractivity relative to vehicle-treated animals was expressed as apercentage; percentage sedation was calculated as 100% minus the percentlocomoter activity.

As shown in FIG. 2 (upper left panel), brimonidine was approximately 60%sedating at a dose 10-fold greater than the 100 μg/kg dose which gave a50% reduction in sulprostone sensitization. Furthermore,dexmeditomidine, shown in the upper right panel of FIG. 2, wascompletely sedating at a dose 10-fold greater than the dose required toproduce a 50% reduction in sensitization score. In contrast, Compound 1administered orally, at a dose of 1 μg/kg, produced a 50% reduction inthe sensitization score (solid line, left axis) with less than 30%sedation (open diamond, right axis) at doses 100-fold and even 1000-foldgreater than the 1 μg/kg dose (see FIG. 2, lower left panel). Similarresults were observed following intraperitoneal administration ofCompound 1. Intraperitoneal administration of Compound 2 also producedmore than a 50% reduction in the sensitization score at 10 μg/kg (solidline, left axis) with less than 30% sedation at a 10-fold greater dose.Thus, Compound 1, which had an extremely low (undetectable) α-1Arelative efficacy, alleviated tactile hypersensitivity withoutconcomitant sedation upon peripheral administration. Similarly, Compound2, which has an α-1A/α-2A potency ratio greater than that ofbrimonidine, also alleviated tactile hypersensitivity withoutconcomitant sedation upon peripheral administration.

In vivo experiments were performed as follows. Sulprostone (CaymanChemical; Ann Arbor, Mich.) was dissolved in dimethyl sulfoxide (DMSO),and brimonidine, phenylephrine, and clonidine were obtained from Sigma(St. Louis, Mo.) and dissolved in saline. Spinal drug injections wereperformed as follows. Mice (20-30 μg) were injected intrathecally asdescribed in Hylden and Wilcox, Eur. J. Pharmacol. 67:313-316 (1980).Briefly, a sterile 30-gauge ½ inch needle attached to a microsyringe wasinserted between the L5 and L6 vertebrae. The mouse was held firmly bythe pelvic girdle in one hand, while the syringe was held in the otherhand at an angle of approximately 20° above the vertebral column. Theneedle was inserted into the tissue to one side of the L6 spinousprocess; into the groove between the spinous and transverse processes.The needle angle was decreased to about 10°, and the needle slowlyadvanced forward into the intervertebral space until a pop was felt andthere was a visible serpentine tail movement. Compounds were slowlyinjected in the subarachnoid space in a volume of 5 μl. Each compoundwas tested at multiple doses. The minimal efficacious dose was used forall subsequent experiments.

Sensitivity to light touch was quantified by scoring the response ofmice to light stroking of their flanks with a small paintbrush, which isnot normally painful. The mice were rated on the following scale onceevery 5 minutes between 15 and 50 minutes post injection: a score of “2”was given to animals showing aggressive escape responses along withsqueaking and biting at the brush; a score of “1” was given to animalsexhibiting mild squeaking with attempts to escape; and a score of “0”was given if the animal showed no response to the light stroking of thepaintbrush. The scores were summed to generate a cumulative score of 0to 16 as described in Minami et al., Pain 57:217-223 (1994). Statisticalcalculations of significance for in vivo studies were done using atwo-tailed Student's t-test.

In sum, these results indicate that α-2A/α-1A adrenergic receptorfunctional selectivity of α-2 agonists in in vitro cell-based functionalassays is associated with lack of sedative activity at the therapeuticdose following systemic or other peripheral dosing. These resultsfurther indicate that Compound 1, which exhibits an α-2A/α-1A adrenergicreceptor in vitro functional selectivity better than the selectivity ofbrimonidine, is a particularly useful α-2 agonist due to the lack of invivo sedative effects at therapeutic doses.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A pharmaceutical composition comprising: a compound represented by

or a pharmaceutically acceptable salt thereof; a pharmaceuticallyacceptable carrier; and at least one additional component selected fromthe group consisting of one or more emulsifying agent, wetting agent,sweetening agent, flavoring agent, tonicity adjuster, preservative,buffer, anti-oxidant and combinations thereof.
 2. The pharmaceuticalcomposition according to claim 1 wherein the pharmaceutically acceptablecarrier is selected from a solid, a semi-solid or a liquid.
 3. Thepharmaceutical composition according to claim 2 wherein the solid isselected from the group consisting of mannitol, lactose, starch,magnesium stearate, sodium saccharin, polyalkylene glycol, talcum,cellulose, glucose, sucrose, magnesium carbonate, and combinationsthereof.
 4. The pharmaceutical composition according to claim 2 whereinthe semi-solid is propylene glycol.
 5. The pharmaceutical compositionaccording to claim 2 wherein the liquid is selected from the groupconsisting of water, distilled water, deionized water, saline, aqueousdextrose, glycerol, ethanol and combinations thereof.
 6. Thepharmaceutical composition according to claim 1 wherein the tonicityadjustor is selected from the group consisting of sodium acetate, sodiumchloride, potassium chloride, mannitol, glycerin, and combinationsthereof.
 7. The pharmaceutical composition according to claim 1 whereinthe preservative is selected from the group consisting of benzalkoniumchloride, chlorobutanol, thimerosal, phenylmercuric acetate,phenylmercuric nitrate, and combinations thereof.
 8. The pharmaceuticalcomposition according to claim 1 wherein the buffer is selected from thegroup consisting of an acetate buffer, a citrate buffers, a phosphatebuffer, a borate buffer, and combinations thereof.
 9. The pharmaceuticalcomposition according to claim 1 wherein the anti-oxidant is selectedfrom the group consisting of sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, andcombinations thereof.
 10. The pharmaceutical composition according toclaim 1 wherein the composition is administered topically.
 11. Thepharmaceutical composition according to claim 1 wherein the compositionis administered orally.
 12. The pharmaceutical composition according toclaim 1 wherein the composition is administered rectally.
 13. Thepharmaceutical composition according to claim 1 wherein the compositionis injected.
 14. A method of alleviating one or more symptom associatedwith anesthesia comprising: administering anesthesia to a patient;discovering the patient is experiencing one or more symptom associatedwith the anesthesia; administering a pharmaceutical compositionaccording to claim 1; and alleviating one or more symptom associatedwith the anesthesia.
 15. The method according to claim 14 wherein one ormore symptom associated with anesthesia is selected from the groupconsisting of nausea, vomiting, shivering, panic, enhancement of memory,enhancement of cognitive processes, and combinations thereof.
 16. Themethod according to claim 15 wherein the alleviation of one or moresymptom associated with anesthesia occurs without concomitant sedation.17. A method of treating a migraine headache comprising: administering apharmaceutical composition according to claim 1 to a patientexperiencing a headache, thereby treating the headache.
 18. A method oftreating a migraine aura headache comprising: administering apharmaceutical composition according to claim 1 to a patientexperiencing a headache migraine aura.